Use of rnai inhibiting parp activity for the manufacture of a medicament for the treatment of cancer

ABSTRACT

The present invention relates to the use of an agent that inhibits the activity of an enzyme that mediates repair of a DNA strand break in the manufacture of a medicament for the treatment of diseases caused by a defect in a gene that mediates homologous recombination.

CROSS REFERENCE

This application claims benefit and is a Division of application Ser.No. 16/598,808, filed Oct. 10, 2019, which claims the benefit ofapplication Ser. No. 16/291,319 filed Mar. 4, 2019, now abandon, whichis a Continuation of application Ser. No. 15/650,459 filed Jul. 14,2017, now abandon, which is a Continuation of application Ser. No.14/322,759 filed Jul. 23, 2014, now abandon, which is a Continuation ofapplication Ser. No. 10/555,507 filed Sep. 15, 2006, now U.S. Pat. No.8,859,562, issued Oct. 14, 2014, which is a 371 application and claimsthe benefit of PCT Application No. PCT/GB2004/003235, filed Jul. 23,2004. This application also claims benefit to GB0317466.1 filed on Jul.25, 2003. These applications are incorporated herein by reference intheir entirety.

This invention relates to the use of an agent that inhibits the activityof an enzyme which mediates the repair of DNA strand breaks in thetreatment of certain forms of cancer in particular breast cancer.

Homologous recombination (HR) has been shown to play an important rolein repair of damage occurring at DNA replication forks in mammaliancells (2). Thus, cells deficient in HR show retarded growth and exhibithigher level of genetic instability. It is believed that geneticinstability due to loss of HR repair in human cancers significantlycontributes to the development of cancer in these cells (1).

Post transcriptional modification of nuclear proteins bypoly(ADP-ribosyl)ation (PARP) in response to DNA strand breaks plays animportant role in DNA repair, regulation of apoptosis, and maintenanceof genomic stability.

Poly(ADP-ribose) Polymerase (PARP-1) is an abundant nuclear protein inmammalian cells that catalyses the formation of poly(ADP-ribose) (PAR)polymers using NAD as substrate. Upon DNA damage, PARP-1 binds rapidlyto a DNA strand break (single strand or double strand) and catalyses theaddition of negatively charged PAR chains to itself (automodification)and other proteins (see [3, 4] for reviews). The binding of PARP-1 toDNA strand breaks is believed to protect DNA lesions from furtherprocessing until PARP-1 is dissociated from the break by the accumulatednegative charge resulting from PAR polymers (5,6).

Although PARP-1 has been implicated in several nuclear processes, suchas modulation of chromatin structure, DNA replication, DNA repair andtranscription, PARP-1 knockout mice develop normally (7). Cells isolatedfrom these mice exhibit a hyper recombination phenotype and geneticinstability in the form of increased levels of SCE, micronuclei andtetraploidy (8-10). Genetic instability may also occur in these PARP-1knockout mice through telomere shortening, increased frequency ofchromosome fusion and aneuploidy (11), although all of these resultscould not be repeated in another set of PARP-1 knock-out mice (12). Inthe former mice knockout, PARP-1 null mutation rescue impaired V(D)Jrecombination in SCID mice (13). These results support the viewsuggested by Lindahl and coworkers that PARP-1 has a protective roleagainst recombination (5). They proposed that binding of PARP-1 to DNAstrand breaks prevents the recombination machinery from recognizing andprocessing DNA lesions or, alternatively, that the negative chargesaccumulated following poly ADP-ribosylation repel adjacentrecombinogenic DNA sequences. Only the latter model is consistent withinhibition of PARP-1 itself and expression of a dominant negative mutantPARP-1, inducing SCE, gene amplification and homologous recombination(HR [14-18]).

Studies based on treating cells with PARP inhibitors or cells derivedfrom PARP-1 or PARP-2 knockout mice indicate that the suppression ofPARP-1 activity increases cell susceptibility to DNA damaging agents andinhibits strand break rejoining (3, 4, 8-11, 19, 20, 47).

Inhibitors of PARP-1 activity have been used in combination withtraditional anti-cancer agents such as radio therapy and chemotherapy(21). The inhibitors were used in combination with methylating agents,topoisomerase poisons and ionising radiations and were found to enhancethe effectiveness of these forms of treatment. Such treatments, however,are known to cause damage and death to non cancerous or “healthy” cellsand are associated with unpleasant side effects.

There is therefore a need for a treatment for cancer that is botheffective and selective in the killing of cancer cells and which doesnot need to be administered in combination with radio or chemotherapytreatments.

The present inventors have surprisingly found that cells deficient inhomologous recombination (HR) are hypersensitive to PARP inhibitors ascompared to wild type cells. This is surprising since PARP-1 knockoutmice live normally thereby indicating that PARP-1 is not essential forlife. Thus, it could not be expected that cells would be sensitive toPARP inhibition.

According to a first aspect of the invention there is provided the useof an agent that inhibits the activity of an enzyme that mediates therepair of DNA strand breaks in the manufacture of a medicament for thetreatment of diseases that are caused by a genetic defect in a gene thatmediates homologous recombination.

In a further aspect the invention provides a method of treatment of adisease or condition in a mammal, including human, which is caused by agenetic defect in a gene which mediates homologous recombination, whichmethod comprises administering to the mammal a therapeutically effectiveamount of an agent which inhibits the activity of an enzyme whichmediates repair of DNA strand breaks or other lesions present atreplication forks.

In a preferred aspect said enzyme is PARP. In a further preferred aspectsaid agent is a PARP inhibitor or an RNAi molecule specific to PARPgene.

In a further preferred aspect, the use is in the treatment of cancer.

Preferably the medicament is a pharmaceutical composition consisting ofthe PARP inhibitor in combination with a pharmaceutically acceptablecarrier or diluent.

The specific sensitivity of HR defective tumours to PARP-1 inhibitionmeans that normally dividing cells in the patient will be unaffected bythe treatment. Treatment of HR defective cancer cells using a PARPinhibitor also has the advantage that it does not need to beadministered as a combination therapy along with conventional radio orchemotherapy treatments thereby avoiding the side effects associatedwith these conventional forms of treatment.

A genetic defect in a gene which mediates homologous recombination maybe due to a mutation in, the absence of, or defective expression of, agene encoding a protein involved in HR.

In a further aspect, the invention further provides the use of a PARPinhibitor in the manufacture of a medicament for inducing apoptosis inHR defective cells.

In another aspect the invention provides a method of inducing apoptosisin HR defective cells in a mammal which method comprises administeringto the mammal a therapeutically effective amount of a PARP inhibitor.

By causing apoptosis in HR defective cells it should be possible toreduce or halt the growth of a tumour in the mammal.

Preferably, the HR defective cells are cancer cells.

Cancer cells defective in HR may partially or totally deficient in HR.Preferably the cancer cells are totally deficient in HR.

The term “cancer” or “tumour” includes lung, colon, pancreatic, gastric,ovarian, cervical, breast or prostate cancer. The cancer may alsoinclude skin, renal, liver, bladder or cerebral cancer. In a preferredaspect, the cancer is in a mammal, preferably human.

The cancer to be treated may be an inherited form of cancer wherein thepatient to be treated has a familial predisposition to the cancer.Preferably, the cancer to be treated is gene-linked hereditary cancer.In a preferred embodiment of the invention the cancer is gene-linkedhereditary breast cancer.

In a preferred aspect, the PARP inhibitor is useful in the treatment ofcancer cells defective in the expression of a gene involved in HR. Geneswith suggested function in HR include XRCC1, ADPRT (PARP-1), ADPRTL2(PARP-2), CTPS, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51,RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, BRCA1, BRCA2, RAD52, RAD54,RAD50, MRE11, NBS1, WRN, BLM, Ku70, Ku80, ATM, ATR, chk1, chk2, FANCA,FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, RADS, FEN-1,Mus81. Eme1, DDS1, BARD (see (2, 3, 5, 22-28) for reviews).

A gene involved in HR may be a tumour suppressor gene. The inventionthus provides for the treatment of cancer cells defective in theexpression of a tumour suppressor gene. Preferably, the tumoursuppressor gene is BRCA1 or BRCA2.

Breast cancer is the most common cancer disease among women in theWestern world today. Certain families have strong predisposition forbreast cancer, which is often owing to an inherited mutation in oneallele of either BRCA1 or BRCA2. However, these patients still maintainone functional allele. Thus, these patients develop normally and have nophenotypic consequence from this mutation. However, in one cell, thefunctional allele might be lost, making this cell cancerous and at thesame time deficient in homologous recombination (HR). This step iscritical for the onset of a tumour (1).

The present inventors have surprisingly found that BRCA2 deficient cellsare 100 times more sensitive to the cytotoxicity of the PARP inhibitor,NU1025, than wild type cells.

Thus in a preferred aspect, the invention provides the use of a PARPinhibitor in the manufacture of a medicament for the treatment of cancercells defective in HR, e.g due to the loss of BRCA1 and/or BRCA2expression.

The cancer cells to be treated may be partially or totally deficient inBRCA1 or BRCA2 expression. BRCA1 and BRCA2 mutations can be identifiedusing multiplex PCR techniques, array techniques (29, 30) or using otherscreens known to the skilled person.

PARP inhibitors useful in the present invention may be selected frominhibitors of PARP-1, PARP-2, PARP-3, PARP-4, tankyrase 1 or tankyrase 2(see 31 for a review). In a preferred embodiment, the PARP inhibitoruseful in the present invention is an inhibitor of PARP-1 activity.

PARP inhibitors useful in the present invention includebenzimidazole-carboxamides, quinazolin-4-[3H]-ones and isoquinolinederivatives (e.g. 2-(4-hydroxyphenyl)benzimidazole-4-carboxamide(NU1085), 8-hydroxy-2-methylquinazolin-4-[3H] one (NU1025);6(5H)phenanthridinone; 3 aminobenzamide; benzimidazole-4-carboxamides(BZ1-6) and tricyclic lactam indoles (TI1-5) [32]. Further inhibitors ofPARP may be identified either by design [33] or the novel FlashPlateassay [34].

The PARP inhibitor formulated as a pharmaceutical composition may beadministered in any effective, convenient manner effective for targetingcancer cells including, for instance, administration by oral,intravenous, intramuscular, intradermal, intranasal, topical routesamong others. Carriers or diluents useful in the pharmaceuticalcomposition may include, but are not limited to saline, buffered saline,dextrose, water, glycerol, ethanol and combinations thereof.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion. The inhibitor may be administered directly to atumour or may be targeted to the tumour via systemic administration.

A therapeutically effective amount of the inhibitor is typically onewhich is sufficient to achieve the desired effect and may vary accordingto the nature and severity of the disease condition, and the potency ofthe inhibitor. It will be appreciated that different concentrations maybe employed for prophylaxis than for treatment of an active disease.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be up to 100 mg/kg,for example from 0.01 mg/kg to 50 mg/kg body weight, typically up to0.1, 0.5, 1.0, 2.0 5.0, 10, 15, 20 or 30 mg/kg body weight. Ultimately,however, the amount of inhibitor administered and the frequency ofadministration will be at the discretion of a physician.

A therapeutic advantage of using PARP inhibitors to treat cancer cellsis that only very low doses are needed to have a therapeutic effect intreating cancer thereby reducing systemic build up of the inhibitors andany associated toxic effects.

A preferred aspect of the invention provides an agent which is aninhibitory RNA (RNAi) molecule.

A technique to specifically ablate gene function is through theintroduction of double stranded RNA, also referred to as inhibitory RNA(RNAi), into a cell which results in the destruction of mRNAcomplementary to the sequence included in the RNAi molecule. The RNAimolecule comprises two complementary strands of RNA (a sense strand andan antisense strand) annealed to each other to form a double strandedRNA molecule. The RNAi molecule is typically derived from exonic orcoding sequence of the gene which is to be ablated.

Preferably said RNAi molecule is derived from the nucleic acid moleculecomprising a nucleic acid sequence selected from the group consistingof: [0040]

a) a nucleic acid sequence as represented by the sequence in FIG. 9, 10,11, 12, 13 or 14 or fragment thereof; [0041]

b) a nucleic acid sequence which hybridises to the nucleic acidsequences of FIG. 9, 10, 11, 12, 13 or 14 and encodes a gene for PARP;[0042]

c) a nucleic acid sequence which comprise sequences which are degenerateas a result of the genetic code to the nucleic acid sequences defined in(a) and (b).

Recent studies suggest that RNAi molecules ranging from 100-1000 bpderived from coding sequence are effective inhibitors of geneexpression. Surprisingly, only a few molecules of RNAi are required toblock gene expression which implies the mechanism is catalytic. The siteof action appears to be nuclear as little if any RNAi is detectable inthe cytoplasm of cells indicating that RNAi exerts its effect duringmRNA synthesis or processing.

More preferably said RNAi molecule according has a length of between 10nucleotide bases (nb)-1000 nb. Even more preferably said RNAi moleculehas a length of 10 nb; 20 nb; 30 nb; 40 nb; 50 nb; 60 nb; 70 nb; 80 nb;90 nb; or 100 bp. Even more preferably still said RNAi molecule is 21 nbin length.

Even more preferably still the RNAi molecule comprises the nucleic acidsequence aaa age cau ggu gga gua uga (PARP-1)

Even more preferably still the RNAi molecule consists of the nucleicacid sequence aag acc aau cuc ucc agu uca ac (PARP-2)

Even more preferably still the RNAi molecule consists of the nucleicacid sequence aag acc aac auc gag aac aac (PARP-3)

The RNAi molecule may comprise modified nucleotide bases.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis.

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1A-1C is a graph demonstrating that HR deficient cells arehypersensitive to the toxic effect caused by inhibition of PARP-1.Colony outgrowth of the Chinese hamster cell lines AA8 (wild-type),irs1SF (deficient in HR[4]), CXR3 (irs1SF complemented with XRCC3 [2]),V79 (wild-type), irs1 (deficient in HR[5]) or irs1X2.2 (irs1complimented with XRCC2 [1]) upon exposure to 3-AB (FIG. 1A), ISQ (FIG.1B) or NU1025 (FIG. 1C). The means (symbols) and standard deviation(bars) of at least three experiments are shown. Colony outgrowth assaywas used;

FIG. 2 is a graph showing cell survival in the presence of PARPinhibitor NU1025 in wt V79 cells, BRCA2 deficient VC-8 cells and VC-8cells complimented with functional BRCA2 gene (VC-8 #13, VC-8+B2).Colony outgrowth assay was used;

FIG. 3 is a histogram showing the percentage of the cells in apoptosisfollowing a 72 hour incubation with NU1025;

FIG. 4A-4C. (FIG. 4A) Western blot analysis of protein lysates isolatedfrom MCF-7 (p53′) or MDA-MB-231 (p53′) breast cancer cells following 48hours transfection with siRNA. (FIG. 4B) Colony outgrowth ofsiRNA-treated MCF-7 cells or (FIG. 4C) MDA-MB-231 cells followingexposure to the PARP inhibitor NU1025. The means (symbols) and standarddeviation (bars) of at least three experiments are shown.

FIG. 5A-5E. BRCA2 deficient cells fail to repair a recombination lesionformed at replication forks by inhibitors of PARP. (FIG. 5A)Visualization of double strand breaks (DSBs) in BRCA2 proficient ordeficient cells following a 24-hour treatment with NU1025 (0.1 mM) bypulse-field gel electrophoresis. Hydroxyurea 2 mM was used as a positivecontrol. (FIG. 5B) Visualisation of .gamma.H2Ax foci in untreatedV-C8+B2 and V-C8 cells. Number of cells containing .gamma.H2Ax foci(FIG. 5C) or RAD51 foci (FIG. 5D) visualised in V-C8+B2 and V-C8 cellsfollowing a 24-hour treatment with NU1025 (10 μM). The means (symbols)and standard errors (bars) of three to nine experiments are shown. (FIG.5E) A suggested model for cell death induced in BRCA2 deficient cells.

FIG. 6A-6C. PARP-1 and not PARP-2 is important in preventing formationof a recombinogenic lesion, causing death in absence of BRCA2. (FIG. 6A)RT-PCR on RNA isolated from SW480SN.3 cells treated with BRCA2, PARP-1and PARP-2 siRNA in combinations as shown for 48 hours. (FIG. 6B)Clonogenic survival following 48-hours depletion of BRCA2, PARP-1 andPARP-2. The means (symbols) and standard deviation (bars) of at leastthree experiments are shown. Two and three stars designate statisticalsignificance in t-test p<0.01 and p<0.001, respectively. (FIG. 6C)Western blot for PARP-1 in SW480SN.3 cells treated with different siRNA.

FIG. 7A-7F. (FIG. 7A) Visualisation of PAR polymers in untreated and(FIG. 7B) thymidine treated V79 cells (5 mM for 24 hours). (FIG. 7C)Percentage cells containing >10 sites of PARP activity followingtreatment with hydroxyurea (0.2 mM) and thymidine (5 mM). At least 300nuclei were counted for each treatment and experiment. (FIG. 7D)Survival of V-C8+B2 cells following co-treatment with hydroxyurea or(FIG. 7E) thymidine and NU1025 (10 μM). (FIG. 7F) The activity of PARPwas measured by the level of free NAD(P)H¹¹, following treatment withMMS, hydroxyurea (0.5 mM) or thymidine (10 mM). The means (symbol) andstandard deviation (error bars) from at least three experiments aredepicted.

FIG. 8A-8F. (FIG. 8A) Visualisation of PAR polymers in untreated V-C8and (FIG. 8B) V-C8+B2 cells. (FIG. 8C) Quantification of percentagecells containing >10 sites of PARP activity in untreated V-C8 andV-C8+B2 cells. (FIG. 8D) Level of NAD(P)H measured in untreated V-C8 andV-C8+B2 cells. Three stars designate p<0.001 in t-test. (FIG. 8E)Visualization of RAD51 and sites of PARP activity in V79 cells followinga 24-hour thymidine treatment (5 mM). (FIG. 8F) A model for the role ofPARP and HR at stalled replication forks.

FIG. 9A-9B is the human cDNA sequence of PARP-1;

FIG. 10 is the human cDNA sequence of PARP-2;

FIG. 11 is the human cDNA sequnce of PARP-3;

FIG. 12A-12B is the human gDNA sequnce of Tankyrase 1;

FIG. 13A-13C is the human mRNA sequnce of Tankyrase 2;

FIG. 14A-14B is the human mRNA sequnce of VPARP.

MATERIALS AND METHODS

Cytotoxicity of PARP Inhibitors to HR-Defective Cells: XRCC2, XRCC3 orBRCA2

Cell Culture

The irs1, irs1X2.1 and V79-4 cell lines were a donation from JohnThacker [40] and the AA8, irs1SF and CXR3 cell lines were provided byLarry Thompson [41].

The VC-8, VC-8+B2, VC-8 #13 were a gift from Malgorzata Zdzienicka [42].All cell lines in this study were grown in Dulbecco's modified Eagle'sMedium (DMEM) with 10% Foetal bovine serum and penicillin (100 U/ml) andstreptomycin sulphate (100 μ·g/mL) at 37° C. under an atmospherecontaining 5% CO2.

Toxicity Assay—Colony Outgrowth Assay

500 cells suspended in medium were plated onto a Petri dish 4 hoursprior to the addition of 3-AB, ISQ or NU1025. ISQ and NU1025 weredissolved in DMSO to a final concentration of 0.2% in treatment medium.7-12 days later, when colonies could be observed, these colonies werefixed and stained with methylene blue in methanol (4 g/l). Coloniesconsisting of more than 50 cells were subsequently counted.

Apoptosis Experiments

0.25.times.10⁶ cells were plated onto Petri dishes and grown for 4 hoursbefore treatment with NU1025. After 72 hours, cells were trypsinized andresuspended with medium containing any floating cells from that sample.The cells were pelleted by centrifugation and resuspended for apoptosisanalysis with FITC-conjugated annexin-V and propidium iodine (PI)(ApoTarget, Biosource International) according to manufacturer'sprotocol. Samples were analysed by flow cytometry (Becton-DickensonFACSort, 488 nm laser), and percentage of apoptotic cells was determinedby the fraction of live cells (PI-negative) bound with FITC-conjugatedannexin-V.

Immunofluorescence

Cells were plated onto coverslips 4 h prior to 24-h treatments asindicated. Following treatments the medium was removed and coverslipsrinsed once in PBS at 37° C. and fixed as described elsewhere [2]. Theprimary antibodies and dilutions used in this study were; rabbitpolyclonal anti PAR (Trevigen; 1:500), goat polyclonal anti Rad51 (C-20,Santa Cruz; 1:200) and rabbit polyclonal anti Rad51 (H-92, Santa Cruz;1:1000). The secondary antibodies were Cy-3-conjugated goat anti-rabbitIgG antibody (Zymed; 1:500), Alexa 555 goat anti-rabbit F(ab′)2 IgGantibody (Molecular Probes; 1:500), Alexa 546 donkey anti-goat IgGantibody (Molecular Probes; 1:500) and Alexa 488 donkey anti-rabbit IgGantibody (Molecular Probes; 1:500). Antibodies were diluted in PBScontaining 3% bovine serum albumin. DNA was stained with 1 μg/ml To Pro(Molecular Probes). Images were obtained with a Zeiss LSM 510 invertedconfocal microscope using planapochromat 63X/NA 1.4 oil immersionobjective and excitation wavelengths 488, 546 and 630 nm. Through focusmaximum projection images were acquired from optical sections 0.50 μmapart and with a section thickness of 1.0 μm. Images were processedusing Adobe PhotoShop (Abacus Inc). At least 300 nuclei were counted oneach slide and those containing more than 10 RAD51 foci or sites of PARPactivity were classified as positive.

PARP Activity Assays

A water-soluble tetrazolium salt (5 mM WST-8) was used to monitor theamount of NAD(P)H through its reduction to a yellow coloured formazandye[43]. 5000 cells were plated in at least triplicate into wells of a96 well plate and cultured in 100 μl normal growth media for 4 h at 37°C.K8 buffer (Dojindo Molecular Technology, Gaithersburg, USA),containing WST-8, was then added either with or without treatment withDNA damaging agents at concentrations indicated. Reduction of WST-8 inthe presence of NAD(P)H was determined by measuring visible absorbance(OD450) every 30 min. A medium blank was also prepared containing justmedia and CK8 buffer. Changes in NAD(P)H levels were calculated bycomparing the absorbance of wells containing cells treated with DNAdamaging agents and those treated with DMSO alone. Alternately relativelevels of NAD(P)H in different cells lines were calculated after 4 hincubation in CK8 buffer.

The ability of NU1025 to inhibit PARP-1 activity was also assayed inpermeabilised cells using a modification of the method of Halldorsson etal [44], and described in detail elsewhere [45]. Briefly: 300 μl ofNU1025-treated (15 min) permeabilised cells were incubated at 26° C.with oligonucleotide (final conc. 2.5 μg/ml), 75imMNAD+[³²P] NAD(Amersham Pharmacia, Amersham, UK) in a total volume of 400 μl. Thereaction was terminated after 5 min by adding ice cold 10% TCA 10% NaPpi for 60 min prior to filtering through a Whatman GF/C filter(LabSales, Maidstone, UK), rinsed 6.times. with 1% TCA 1% NaPPi, left todry and incorporated radioactivity was measured to determine PARP-1activity. Data are expressed as pmol NAD incorporated/10⁶ cells byreference to [³²P] NAD standards.

Pulse-Field Gel Electrophoresis

1.5.times.10⁶ cells were plated onto 100 mm dishes and allowed 4 h forattachment. Exposure to drug was for 18 h after which cells weretrypsinsied and 10⁶ cells melted into each 1% agarose insert. Theseinserts were incubated as described elsewhere (8) and separated bypulse-field gel electrophoresis for 24 h (BioRad; 120° angle, 60 to 240s switch time, 4 V/cm). The gel was subsequently stained with ethidiumbromide for analysis.

siRNA Treatment

Predesigned BRCA2 SMARTpool and scrambled siRNAs were purchased(Dharmacon, Lafayette, Colo.). 10000 cells seeded onto 6 well plates andleft over night before transfected with 100 nM siRNA usingOligofectamine Reagent (Invitrogen) according to manufacturersinstructions. Cells were then cultured in normal growth media for 48 hprior to trypsinisation and replating for toxicity assays. Suppressionof BRCA2 was confirmed by Western blotting (as described previously[46]) of protein extracts treated with siRNA with an antibody againstBRCA2 (Oncogene, Nottingham, UK).

EXAMPLES

Homologous Recombination Deficient Cells are Hypersensitive to PARP-1Inhibition

To investigate the involvement of HR in cellular responses to inhibitionof PARP-1, the effects of PARP-1 inhibitors on the survival of HR repairdeficient cell lines were studied. It was found that cells deficient inHR (i.e., irs1SF which is defective in XRCC3 or irs1 which is defectivein XRCC2 [see Table 1] were very sensitive to the toxic effect of3-aminobenzamide (3-AB) and to two more potent inhibitors of PARP-1:1,5-dihydroxyisoquinoline (ISQ; [37]) or 8-hydroxy-2-methylquinazolinone(NU1025 [38, 39]) (FIG. 1). The sensitivity in irs1SF cells to 3-AB, ISQor NU1025 was corrected by the introduction of a cosmid containing afunctional XRCC3 gene (CXR3). Similarly, the sensitivity in irs1 cellsto 3-AB, ISQ or NU1025 was corrected by the introduction of a cosmidcontaining a functional XRCC2 gene (irs1X2.2).

BRCA2 Deficient Cells are Hypersensitive to PARP-1 Inhibition

The survival of BRCA2 deficient cells (VC8) and wild type cells (V79Z)in the presence of inhibitors of PARP-1 was investigated. It was foundthat VC8 cells are very sensitive to the toxic effect of NU1025 (FIG.2). The sensitivity in VC8 cells was corrected by the introduction of afunctional BRCA2 gene either on chromosome 13 (VC8 #13) or on anoverexpression vector (VC8+B2). This result demonstrates that thesensitivity to PARP-1 inhibitors is a direct consequence of loss of theBRCA2 function.

To investigate if inhibition of PARP-1 triggers apoptosis in BRCA2deficient cells, the level of apoptosis 72 hours following exposure toNU1025 was investigated. It was found that NU1025 triggered apoptosisonly in VC8 cells, showing that loss of PARP-1 activity in BRCA2deficient cells triggers this means of death (FIG. 3).

BRCA2 Deficient Breast Cancer Cells are Hypersensitive to PARP-1Inhibition

It was examined whether the MCF7 (wild-type p53) and MDA-MB-231 (mutatedp53) breast cancer cell lines displayed a similar sensitivity to NU1025upon depletion of BRCA2. It was found that PARP inhibitors profoundlyreduced the survival of MCF7 and MDA-MB-231 cells only when BRCA2 wasdepleted with a mixture of BRCA2 siRNA (FIG. 4). This shows that BRCA2depleted breast cancer cells are sensitive to PARP inhibitors regardlessof p53 status.

BRCA2 Deficient Cells Die from PARP-1 Inhibition in Absence of DNADouble-Strand Breaks (DSBs) but in Presence of yH2Ax

HR is known to be involved in the repair of DSBs and other lesions thatoccur during DNA replication [2]. To determine whether the sensitivityof BRCA2 deficient cells is the result of an inability to repair DSBsfollowing NU1025 treatment, the accumulation of DSBs in V79 and V-C8cells was measured following treatments with highly toxic levels ofNU1025. It was found that no DSBs were detectable by pulsed field gelelectrophoretic analysis of DNA obtained from the treated cells (FIG.5A), suggesting that low levels of DSBs or other recombinogenicsubstrates accumulated following PARP inhibition in HR deficient cells,which trigger .gamma.H2Ax FIG. 5B). The reason why BRCA2 deficient cellsdie following induction of these recombinogenic lesions is likely to bedue to an inability to repair such lesions. To test this, the ability ofBRCA2 deficient V-C8 cells and BRCA2 complimented cells to form RAD51foci in response to NU1025 was determined. It was found that RAD51 fociwere indeed induced in V-C8+B2 cells following treatment with NU1025(statistically significant in t-test p<0.05; FIG. 5D). This indicatesthat the recombinogenic lesions trigger HR repair in these cellsallowing them to survive. In contrast, the BRCA2 deficient V-C8 cellswere unable to form RAD51 foci in response to NU1025 treatment (FIG. 5D)indicating no BR, which would leave the recombinogenic lesionsunrepaired and thus cause cell death.

PARP-1 and not PARP-2 is Important in Preventing Formation of aRecombinogenic Lesion

There are two major PARPs present in the nucleus in mammalian cells,PARP-1 and PARP-2 and all reported PARP inhibitors inhibit both. Inorder to distinguish which PARP was responsible for the effect, wetested if the absence of PARP-1 and/or PARP-2 results in accumulation oftoxic lesions, by depleting these and BRCA2 with siRNA in human cells(FIG. 6a ). We found that the clonogenic survival was significantlyreduced when both PARP-1 and BRCA2 proteins were co-depleted from humancells (FIG. 6b ). Depletion of PARP-2 with BRCA2 had no effect on theclonogenic survival and depletion of PARP-2 in PARP-1 and BRCA2 depletedcells did not result in additional toxicity. These results suggest thatPARP-1 and not PARP-2 is responsible for reducing toxic recombinogeniclesions in human cells. The cloning efficiency was only reduced to 60%of control in PARP-1 and BRCA2 co-depleted cells, while no HR deficientcells survived treatments with PARP inhibitors. This is likely to dowith incomplete depletion of the abundant PARP-1 protein by siRNA (FIG.6c ), which might be sufficient to maintain PARP-1 function in some ofthe cells.

PARP-1 is Activated by Replication Inhibitors

HR is also involved in repair of lesions occurring at stalledreplication forks, which may not involve detectable DSBs [2]. To test ifPARP has a role at replication forks, PARP activation in cells treatedcells with agents (thymidine or hydroxyurea) that retard or arrest theprogression of DNA replication forks was examined. Thymidine depletescells of dCTP and slows replication forks without causing DSBs.Hydroxyurea depletes several dNTP and block the replication fork, whichis associated with the formation of DSBs at replication forks [2]. Bothof these agents potently induce HR [2]. V79 hamster cells treated for 24hours with thymidine or hydroxyurea were stained for PAR polymers. Thisrevealed a substantial increase in the number of cells containing sitesof PARP activity (FIG. 7C). This result suggests a function for PARP atstalled replication forks. It was also shown that inhibition of PARPwith NU1025 enhances the sensitivity to thymidine or hydroxyurea inV-C8+B2 cells (FIG. 7D,E). This result suggests that PARP activity isimportant in repair of stalled replication forks or alternatively thatit prevents the induction of death in cells with stalled replicationforks.

PARP is rapidly activated at DNA single-strand breaks (SSB) and attractsDNA repair enzymes [3-6]. Methylmethane sulphonate (MMS) causesalkylation of DNA, which is repaired by base excision repair. PARP israpidly activated by the SSB-intermediate formed during this repair,which depletes the NAD(P)H levels (FIG. 7F). We found that theactivation of PARP and reduction of NAD(P)H levels is much slowerfollowing thymidine or hydroxyurea treatments. This slow PARP activationcan be explained by the indirect action of thymidine and hydroxyurea andthe time required to accumulate stalled replication forks as cells enterthe S phase of the cell cycle.

PARP-1 and HR have Separate Roles at Stalled Replication Forks

The number sites of PARP activity in untreated BRCA2 deficient V-C8cells was determined. It was found that more V-C8 cells contain sites ofPARP activity compared to V-C8+B2 cells (FIG. 8A,B,C). Also, the V-C8cells have lower free NAD(P)H levels than the corrected cells (FIG. 8D),as a likely result of the increased PARP activity. Importantly thesesites of PARP activity do not overlap with RAD51 foci (FIG. 8E).

The results herein suggest that PARP and HR have separate roles in theprotection or rescue of stalled replication forks (FIG. 8F). A loss ofPARP activity can be compensated by increased HR while a loss of HR canbe compensated by increased PARP activity. However, loss of both thesepathways leads to accumulation of stalled replication forks and todeath, as in the case of PARP inhibited BRCA2 deficient cells.

As shown in the model outlined in FIG. 8F PARP and HR have complementaryroles at stalled replication forks. (i) Replication forks may stall whenencountering a roadblock on the DNA template. In addition, they may alsostall temporarily, due to lack of dNTPs or other replication co-factors.(ii) PARP binds stalled replication forks or otherreplication-associated damage, triggering PAR polymerization. Resultingnegatively charged PAR polymers may protect stalled replication forks,by repelling proteins that normally would process replication forks(e.g., resolvases), until the replication fork can be restoredspontaneously when dNTPs or other co-factors become available.Alternatively, PAR polymers or PARP may attract proteins to resolve thereplication block by other means. (iii) In absence of PARP activity, HRmay be used as an alternative pathway to repair stalled replicationforks. This compensatory model explains the increased level of HR andRAD51 foci found in PARP deficient cells³⁻⁵ and higher PARP activityfound in HR deficient cells (i.e. V-C8). Spontaneous replicationblocks/lesions are only lethal in the absence of both PARP and HR.

TABLE 1 Genotype and origin of cell lines used in this study. Cell lineGenotype Defect Origin Reference AA8 Wt Wt CHO [41] irs1SF XRCC3⁻XRCC3⁻, deficient AA8 [41] in HR CXR3 XRCC3⁻ + Wt irs1SF [41] hXRCC3V79-4 Wt Wt V79 [40] irs1 XRCC2⁻ XRCC2⁻, deficient V79-4 [40] in HRirs1X2.2 XRCC2⁻ + Wt irs1 [40] hXRCC2 V79-Z Wt Wt V79 [42] VC8 BRCA2⁻BRCA2⁻, deficient V79-Z [42] in HR VC8#13 BRCA2⁻ + Wt VC8 [42] hBRCA2VC8 + B2 BRCA2⁻ + Wt VC8 [42] hBRCA2

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1. Use of an agent that inhibits the activity of an enzyme that mediatesrepair of a DNA strand break in the manufacture of a medicament for thetreatment of diseases caused by a defect in a gene that mediateshomologous recombination.
 2. The use as claimed m claim 1 wherein theenzyme is poly(ADP-ribose) polymerase (PARP).
 3. The use as claimed inclaim 2 wherein the agent is a PARP inhibitor.
 4. The use as claimed inclaim 3 wherein the PARP inhibitor is selected from the group consistingof PARP-1, PARP-2, PARP-3, PARP-4, tankyrase 1 and tankyrase
 2. 5. Theuse as claimed in claim 4 wherein the PARP is PARP-1.
 6. The use asclaimed in claim 1 or claim 2 wherein the agent is an RNAi moleculespecific to a PARP gene.
 7. The use as claimed in claim 6 wherein theRNAi molecule is derived from a nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of: a) anucleic acid sequence as represented by the sequence in FIG. 9, 10, 11,12, 13 or 14, or a fragment thereof; b) a nucleic acid sequence whichhybridises to the nucleic acid sequences of FIG. 9, 10, 11, 12, 13 or14, and encodes a gene for PARP; or c) a nucleic acid sequence whichcomprises sequences which are degenerate as a result of the genetic codeto the nucleic acid sequences defined in (a) and (b).
 8. The use asclaimed in claim 6 or 7 wherein the RNAi molecule comprises the nucleicacid sequence aaa agc cau ggu gga gua uga.
 9. The use as claimed inclaim 6 or 7 wherein the RNAi molecule consists of the nucleic acidsequence aag acc aau cuc ucc agu uca ac.
 10. The use as claimed in claim6 or 7 wherein the RNAi molecule consists of the nucleic acid sequenceaag acc aac auc gag aac aac.
 11. The use as claimed in any precedingclaim wherein the defect is a mutation in a gene encoding a proteininvolved in HR.
 12. The use as claimed in any of claims 1 to 10 whereinthe defect is the absence of a gene encoding a protein involved in HR.13. The use as claimed in any of claims 1 to 10 wherein the defect is inthe expression of a gene encoding a protein involved in HR.
 14. The useas claimed in any preceding claim wherein the gene that mediates HR isselected from the group consisting of XRCC1, ADPRT (PARP-1), ADPRTL2(PARP-2), CTPS, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51,RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, BRCA1, BRCA2, RAD52, 20RAD54, RAD50, MRE11, NBS1, WRN, BLM, Ku70, Ku80, ATM, ATR, chk1, chk2,FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, RADS,FEN-1, Mus81, Eme1, DDS1 and BARD.
 15. The use as claimed in anypreceding claim in the treatment of cancer.
 16. The use as claimed inclaim 15 wherein the cancer is selected from the group consisting oflung, colon, pancreatic, gastric, ovarian, cervical, breast and prostatecancer.
 17. The use as claimed in claim 15 or 16 wherein the cancer isin a human.
 18. The use as claimed in any of claims 15 to 17 wherein thecancer is gene-linked hereditary cancer.
 19. The use as claimed in claim18 wherein the cancer is breast cancer.
 20. The use as claimed in any ofclaims 15 to 19 wherein the cancer cells to be treated are defective inBRCA1 expression.
 21. The use as claimed in any of claims 15 to 19wherein the cancer cells to be treated are defective in BRCA2expression.
 22. The use as claimed in claim 20 or 21 wherein the cancercells are partially deficient in BRCA1 and/or BRCA2 expression.
 23. Theuse as claimed in claim 20 or 21 wherein the cancer cells are totallydeficient in BRCA1 and/or BRCA2 expression.
 24. The use as claimed inany preceding claim wherein the gene that mediates HR is a tumoursuppressor gene.
 25. The use as claimed in claim 24 wherein the tumoursuppressor gene is BRCA1.
 26. The use as claimed in claim 24 wherein thetumour suppressor gene is BRCA2.
 27. Use of a PARP inhibitor in themanufacture of a medicament for inducing apoptosis in HR. defectivecells.
 28. The use as claimed in claim 27 wherein the HR. defectivecells are cancer cells.
 29. The use as claimed in claim 28 wherein thecancer cells defective in HR are partially deficient in HR.
 30. The useas claimed in claim 28 wherein the cancer cells defective in HR aretotally deficient in HR.
 31. A method of treatment of a disease orcondition in a mammal, including human, which is caused by a geneticdefect in a gene that mediates homologous recombination, which methodcomprises administering to the mammal a therapeutically effective amountof an agent that inhibits the activity of an enzyme that mediates repairof DNA strand breaks.
 32. A method of inducing apoptosis in HR defectivecells in a mammal which method comprises administering to the mammal atherapeutically effective amount of a PARP inhibitor.